Cobalamin (Cbl) or vitamin B.sub.12 deficiency, usually the result of disruption of the absorption of cobalamin, can lead to life-threatening hematological and neuropsychiatric abnormalities. Accurate and early diagnosis of cobalamin deficiency is important since proper treatment with cobalamin results in complete reversal of the hematologic symptoms. Early diagnosis is especially important in order to avoid potentially incapacitating, irreversible neurologic damage. Administration of exogenous cobalamin always stops the progression of neuropsychiatric abnormalities, almost always leads to significant improvements in such symptoms and frequently leads to their complete correction. Early diagnosis is often difficult since the clinical signs of Cbl deficiency can result from a variety of other disorders. It has generally been taught that Cbl deficiency should be suspected in individuals with significant anemia, displayed for example in decreased hematocrit or hemoglobin, with macrocytic red blood cells (i.e., mean cell volume (MCV) generally greater than 100 fl), or in individuals having the neurologic symptoms of peripheral neuropathy and/or ataxia. Anemia associated with Cbl deficiency has been described as typically severe with hemoglobin .ltoreq.8 g % or hematocrit &lt;25% and the size of the red blood cells is described as greatly increased to levels &gt;110 fl. (See, for example, Babior and Bunn (1983) in Harrison's Principles of Internal Medicine (Petersdorf et al., eds.) McGraw-Hill Book Co., New York; Lee and Gardner (1984) In Textbook of Family Practice, 3rd Ed. (Rakel, ed.) Saunders & Co., Philadelphia). While it was well recognized that individuals with Cbl deficiency could display neurologic disorders in the absence of anemia, such situations were believed to be exceptional and rare (Beck (1985) in Cecil Textbook of Medicine, 17th Ed. (Wyngaarden and Smith, eds.) W. B. Saunders, Philadelphia, p. 893-900; Babior and Bunn (1987) in Harrison's Principles of Internal Medicine, 11th Ed. (Braunwald et al., eds.) McGraw-Hill, New York pp.1498-1504; Walton (1985) Brain's Diseases of the Nervous System, 9th Ed. Oxford University Press, Oxford, UK). The neurologic symptoms of Cbl deficiency have often been considered to be late manifestations of the disease most typically occurring after the onset of anemia or if they occur first, are soon to be followed with onset of anemia (Woltmann (1919) Am. J. Med. Sci. 157:400-409; Victor and Lear (1956) Am. J. Med. 20:896-911). These symptoms have also been thought to often result from inappropriate therapy with folic acid (Conley and Krevans (1951) New Eng. J. Med. 245:529-531).
Lindenbaum et al. (1988) New Eng. J. Med. 318:1720-1728 have recently reported that neuropsychiatric disorders resulting from Cbl deficiency occur commonly in the absence of anemia and elevated MCV. They reported that a large percentage (28%) of the Cbl deficient patients displaying neuropsychiatric abnormalities in their study had no anemia or macrocytosis or had only minor hematologic abnormalities. Further, they reported that a much wider spectrum of neuropsychiatric symptoms can result from Cbl deficiency, see also Lindenbaum et al. (1988) Laboratory Management 26:41-44.
The serum cobalamin assay has been essentially the only laboratory assay generally available for use in determining if a patient is Cbl deficient. Presently preferred cobalamin assays are radiodilution assays which use pure or purified intrinsic factor as the binding protein (see: Kolhouse et al. (1978) New Eng. J. Med. 299:785-792). This assay has been criticized as frequently giving low Cbl values in patients who lack any evidence of Cbl deficiency. It has been suggested (Schilling et al. (1983) Clin. Chem. 29:582-583) that this assay may frequently give false positives showing low serum Cbl levels in individuals who are not Cbl deficient.
It has long been known that methylmalonic acid (MMA) is excreted in increased amounts in the urine of most patients with Cbl deficiency (see, for example, Cox and White (1962) Lancet ii:853-856; Norman et al. (1982) Blood 59:1128-1131). In Cbl deficiency, reduced levels of adenosyl-Cbl result in decreased activity of L-methylmalonyl-coenzyme A (CoA) mutase and a concomitant increase in intracellular levels of L-methylmalonyl-CoA. D-methylmalonyl-CoA, resulting from transformation of the L-isomer by D,L-methylmalonyl-CoA racemase (Stabler et al. (1985) Arch. Biochem. Biophys. 241:252-264), is cleaved to CoA and MMAby D-methylmalonyl-CoA hydrolase which has been recently characterized (Kovachy et al. (1983) J. Biol. Chem. 258:11415-11421). MMA is then released into blood in unknown amounts and is excreted in the urine. About 70% of the MMA in blood is metabolized to unknown products via as yet undefined pathways and only about 30% is excreted in the urine. Although it has been suggested that Cbl deficiency can be detected by urine MMA analysis (Norman et al. (1982) Blood 59:1128-1131), in practice urine MMA has rarely been measured and has been deemed rarely necessary in patients suspected of being Cbl deficient, since it has been taught that accurate diagnosis can be based on the presence and degree of anemia and macrocytosis and by the measurement of serum cobalamin levels (see: Beck (1983) in Hematology, 3rd Ed. (Williams et al., eds.) McGraw-Hill, New York, pp. 434-465, Beck (1985) supra).
Recently, a comparison of MMA levels in urine and serum of normal subjects and Cbl deficient subjects has become possible with the development of a sensitive capillary gas chromatography-mass spectrometry method for quantitation of MMA and other dicarboxylic acids (Marcell et al. (1985) Anal. Biochem. 158:58-66; Stabler et al. (1986) J. Clin. Invest. 77:1606-1612; Lindenbaum et al. (1988) New Eng. J. Med. supra; Allen et al. U.S. Patent application Ser. No. 933,553, filed Nov. 20, 1986, which is incorporated by reference herein). Prior to these reports MMA had not been detected in serum of normal subjects. Levels of serum MMA of 26,000 to 340,000 ng/ml had previously been reported in patients with inborn errors of metabolism involving the synthesis of adenosyl-Cbl or the adenosyl-Cbl-dependent enzyme, L-methylmalonyl-CoA mutase (see: Rosenberg (1983) in The Metabolic Basis of Inherited Disease (Goldstein and Brown, eds.) McGraw-Hill, New York p.474-497). Urine MMA levels in these patients were reported to be 10- to 100-fold higher than serum levels. Marcell et al. reported that MMA in the serum and urine of normal subjects ranged from 19-76 ng/ml and 270-7190 ng/ml, respectively. Stabler et al. reported that serum MMA levels of clinically confirmed Cbl deficient patients ranged from 55 to 22,300 ng/ml, with 69 of 73 of such patients having serum MMA levels above the normal range. It was also reported that there was a positive correlation between serum MMA levels and the presence of neurologic abnormalities in these patients. Lindenbaum et al. reported that serum MMA levels were elevated above normal levels in 36 of 37 Cbl deficient patients who displayed neuropsychiatric abnormalities in the absence of anemia or other severe hematologic abnormalities. Further, it is suggested that high serum MMA levels which return to normal after cobalamin therapy provide a useful confirmation of the presence of Cbl deficiency.